The invention relates to the fields of screening assays and compounds and methods for treating cancer and other proliferative diseases.
The regulation of protein expression can occur at a number of levels: transcriptional, post-transcriptional, or post-translational. The modulation of protein expression is often critical for the treatment of disease. To this end, one area of research has been directed at developing small molecules that regulate transcription factors. Another approach to modulating protein expression is achieved by targeting the RNA encoding the target protein using antisense technology.
Another manner in which protein expression is regulated is through modulating translation efficiency. In eukaryotes, translational regulation usually occurs at the initiation step. Therefore, cis-elements in the 5xe2x80x2 untranslated region (UTR) are important for overall regulation of protein synthesis. There is also a growing body of evidence that supports the role of the poly(A) tail and elements in the 3xe2x80x2 UTR in regulating initiation of translation
HER2 overexpression occurs in a number of primary human tumors, including 25-30% of breast and ovarian carcinomas, and is associated with an adverse prognosis and rapid tumor growth. HER2 is regulated at both the transcriptional and post-transcriptional level. For example, it has been previously shown that HER2 mRNA is translated more efficiently in transformed cells than in primary cells. Therefore, therapeutics that decrease HER2 polypeptide levels within a cell would be valuable as drugs for the treatment of conditions such as cancer and other proliferative diseases.
In addition, there is a need for the identification of compounds that can be used to treat cancer and proliferative diseases, regardless of the mechanism of action.
The invention features screening assays for compounds that are potentially useful for treating or preventing a proliferative disease. In addition, the invention features compounds identified by the assays of the invention. The invention further features compounds structurally related to those identified by the screening assays. Finally, the invention features methods of treating or preventing a proliferative disease using the compounds of the invention.
Accordingly, in one aspect, the invention features a method of identifying a compound for treating or preventing a proliferative disease. This method includes the steps of (a) providing a cell, e.g., a mammary cell, a gastric cell, an ovarian cell, a bladder cell, a lung cell, a salivary gland cell, or a cancer cell, including a nucleic acid molecule containing a HER2 regulatory element operatively linked to a reporter coding sequence and stably integrated into a chromosome of the cell; (b) contacting the cell with a candidate compound; and (c) assaying for the effectiveness of said candidate compound for the treatment or prevention of said proliferative disease. In one embodiment, in step (a), the method further includes adding an RNA binding protein to the cell, under conditions that allow interaction between the nucleic acid molecule and the RNA binding protein. The interaction in step (a) between said nucleic acid molecule and said RNA binding protein may also be mediated by at least one polypeptide. The nucleic acid molecule including a HER2 regulatory sequence operatively linked to a reporter coding sequence may further include one or more additional regulatory elements. The HER2 regulatory element may, for example, include a HER2 5xe2x80x2 UTR, a HER2 3xe2x80x2 UTR, or a HER2 5xe2x80x2 UTR and a HER2 3xe2x80x2 UTR. The HER2 5xe2x80x2 UTR is, for example, SEQ ID NO: 1, and the HER2 3xe2x80x2 UTR is, for example, SEQ ID NO: 2.
In one embodiment, the assaying in step (c) is for a change in the level of translation efficiency of the reporter coding sequence relative to a cell not exposed to the candidate compound, and a modulation in the level of translation efficiency of said reporter coding sequence indicates a compound that modulates the translation efficiency of HER2. The modulation may be an increase or decrease in the level of translation efficiency of HER2. The reporter coding sequence is, for example, a luciferase, a xcex2-galactosidase, or a green fluorescent protein reporter sequence. In one embodiment, the translation efficiency is assayed by polysomal distribution analysis, followed by detection of the level of reporter coding RNA expression. In another embodiment, the translation efficiency is assayed by detection of the level of reporter coding protein expression or activity.
In another embodiment, the assaying in step (c) is for toxicity of the candidate compound to the cell or the compounds ability to inhibit cell growth, relative to a cell not exposed to the compound. Step (c) may further include comparing the toxicity or growth inhibition of the candidate compound to the cell to the toxicity or growth inhibition of the compound to a reference cell. The toxicity or growth inhibition of a compound to a reference cell may be, for example, representative of the toxicity or growth inhibitory effect of the compound to the general cell population.
In another aspect, the invention features compounds of the formula: 
or a pharmaceutically acceptable salt thereof, wherein U, V, Y, and Z are independently C, CH, N, O, or S; X is independently H, Hal, lower alkyl, OR5, SR6, or NR7R8, where R5, R6, R7, and R8 are independently H or lower alkyl, and n is 1, 2, 3, or 4; R1 and R2 are independently H, Me, or Et, or R1 and R2 together are (CR9R10)m, where R9 and R10 are independently H, lower alkyl, CH2OR11, (CH2)oNR12R13, or OR14, where R11, R12, R13, and R14 are H, lower alkyl, aryl, or alkaryl, m is 2, 3, 4, or 5, and o is 0 or 1; and Ar is an unsubstituted or substituted carbocyclic or heterocyclic aromatic ring or fused ring system. In one embodiment, U, V, Y, and Z are C or CH.
In another embodiment, the compound is of the formula: 
where V, Y, and Z are independently N, CH, or C; B is N, CH, or CR4, where R4 is F or lower alkyl; X is independently H, Hal, lower alkyl, OR5, SR6, or NR7R8, where R5, R6, R7, and R8 are independently H or lower alkyl, and n is 1, 2, 3, or 4; R1 and R2 are independently H, Me, or Et, or R1 and R2 together are (CR9R10)m, where R9 and R10 are independently H, lower alkyl, CH2OR11, (CH2)oNR12R13, or OR14, where R11, R12, Rxe2x80x94, and R14 are independently H, lower alkyl, aryl, or alkaryl, m is 2, 3, 4, or 5, and o is 0 or 1; and R3 is H, lower alkyl, F, OR15, where R15is H, lower alkyl, or aralkyl, or a substituent of the structure: 
where R16 is NH, O, or S; R17 is H, lower alkyl, or aralkyl; and W is lower alkyl, lower alkenyl, lower alkynyl, aryl, heteroaryl, alkaryl, OR18, or NR19R20, where R18, R19, and R20 are independently H, lower alkyl, lower alkenyl, lower alkynyl, aryl, alkaryl, aralkyl, or (CH2)kCH2OR21, where R21 is H or lower alkyl, and k is 1, 2, 3, or 4. In various embodiments, at least one of B, V, Y, and Z is N. In various other embodiments, when Xn is H, V, Y, and Z are C or CH, R1 and R2 together are (CH2)4, B is CR4, and R4 is F, R3 is not F or H; when Xn is H, V, Y, and Z are C or CH, R1 and R2 together are (CH2)4, B is CR4, and R4 is Me, R3 is not H; when Xn is H, V, Y, and Z are C or CH, R1 and R2 together are (CH2)4, B is CR4, and R4 is H, R3 is not H or F; when Xn is H, V, Y, and Z are C or CH, B is CR4, and R1, R2, and R4 are Me, R3 is not H; when Xn is H, V, Y, and Z are C or CH, R1 and R2 are Me, B is CR4, and R4 is F, R3 is not F; when Xn is H, V, Y, and Z are C or CH, R1 and R2 together are (CH2)4, B is CH, W is methyl, and R16 is O, R17 is not H; when Xn is H, V, Y, and Z are C or CH, R1 and R2 are methyl, B is CH, W is methyl, and R16 is O, R17 is not H; when Xn is H, V, Y, and Z are C or CH, B is CH, and R1 and R2 are Me or R1 and R2 together are (CH2)5, R3 is not methoxy; when Xn is H, V, Y, and Z are C or CH, B is CH, and R1 and R2 together are (CH2)5, R3 is not F; and Xn is H, V, Y, and Z are C or CH, B is CH, and R1 and R2 together are (CH2)5, R3 is not ethoxy. Exemplary compounds of the above embodiments include: 
Compounds of the invention may also be of the formula: 
where W is H, lower alkyl, aryl, Hal, or OR5, where R5 is H or lower alkyl; X is independently H, Hal, lower alkyl, OR6, SR7, or NR8R9, where R6, R7, R8, and R9 are independently H or lower alkyl, and n is 1, 2, 3, or 4; Y is NH, O, or S; Z is O or NR10, where R10 is H, lower alkyl, or lower alkenyl; R1 and R2 are independently H, Me, or Et, or R1 and R2 together are (CR11R12)m, where R11 and R12 are independently H, lower alkyl, CH2OR13, (CH2)oNR14R15, or OR16, where R13, R14, R15, and R16 are H, lower alkyl, aryl, or alkaryl, m is 2, 3, 4, or 5, and o is 0 or 1; R3 is H or lower alkyl; and R4 is H, lower alkyl, lower alkenyl, lower alkynyl, aryl, alkaryl, or (CH2)kCH2OR17, where R17 is H or lower alkyl, and k is 1, 2, 3, or 4. In one embodiment, W is H. Exemplary compounds of these embodiments include: 
In another embodiment, the compounds are of the formula: 
where W is H, lower alkyl, aryl, Hal, or OR5, where R5 is H or lower alkyl; X is independently H, Hal, lower alkyl, OR6, SR7, or NR8R9, where R6, R7, R8, and R9 are independently H or lower alkyl, and n is 1, 2, 3, or 4; Y is NH, O, or S; Z is O or NR10, where R10 is H, lower alkyl, or lower alkenyl; R1 and R2 are independently H, Me, or Et, or R1 and R2 together are (CR11R12)m, where R11 and R12 are independently H, lower alkyl, CH2OR13, (CH2)oNR14R15, or OR16, where R13, R14, R15, and R16 are H, lower alkyl, aryl, or alkaryl, m is 2, 3, 4, or 5, and o is 0 or 1; R3 is H or lower alkyl; and R4 is H, lower alkyl, lower alkenyl, lower alkynyl, aryl, alkaryl, or (CH2)kCH2OR17, where R17 is H or lower alkyl, and k is 1, 2, 3, or 4. An exemplary compound is: 
Compounds of the invention also include compounds of the formula: 
where Y and Z are independently C, CH, or N; B is N or CR4, where R4 is F, lower alkyl, or H; X is in dependently H, Hal, lower alkyl, OR5, SR6, or NR7R8, where R5, R6, R7, and R8 are independently H or lower alkyl, and n is 1, 2, 3, or 4; R1 and R2 are independently H, Me, or Et, or and R1 and R2 together are (CR9R10)m, where R9 and R10 are independently H, lower alkyl, CH2OR11, (CH2)oNR12R13, or OR14, where R11, R12, R13, R14 are H, lower alkyl, aryl, or alkaryl, m is 2, 3, 4, or 5, and o is 0 or 1; and R3 is a substituent structure of the structure: 
where R16 and R17 are independently H, lower alkyl, alkaryl, or aryl. Examples include: 
In another embodiment, the compounds are of the formula: 
where X and Y are independently N or CH; R1 is H, lower alkyl or aralkyl; R2 is lower alkyl, or aralkyl; R3 and R4 are independently H or OR5, where R5 is H, lower alkyl, or NR6R7, where R6 and R7 are independently H or lower alkyl. Exemplary compounds of this embodiment include: 
The invention features compounds of the formula: 
where X and Y are independently N or CH; A is O or S; R1 is H, lower alkyl, or aralkyl; R2 and R3 are independently lower alkyl or aralkyl; and R4 and R5 are independently H or OR6, where R6 is H, lower alkyl, or NR7R8, where R7 and R8 are independently H or lower alkyl.
The invention also features compounds of the formula: 
where X, Y, and Z are independently N or CH; R1 is lower alkyl or aralkyl; and R9 and R10 are independently H, OR14, where R14 is H, lower alkyl, or NR12R13, where R12 and R13 are independently H or lower alkyl.
The invention further features compositions including any one or more compounds of the invention and a pharmaceutically acceptable carrier. Exemplary compounds for such compositions include: 
Any of the compounds of the invention may also be in the form of a pharmaceutically acceptable salt.
In another aspect, the invention features methods for treating or preventing a proliferative disease, such as cancer, in a subject, e.g., a mammal or human. The methods include administering to the subject a therapeutically effective amount of a chemical compound of the invention. The compound may be in a pharmaceutically acceptable carrier. The therapeutically effective amount is, for example, a dosage sufficient to modulate HER2 polypeptide expression.
By xe2x80x9calkarylxe2x80x9d is meant an alkyl, alkenyl, or alkynyl radical substituted with an aryl group.
By xe2x80x9caralkylxe2x80x9d is meant an aryl radical substituted with an alkyl, alkenyl, or alkynyl group.
By xe2x80x9carylxe2x80x9d is meant an aromatic carbocyclic or heterocyclic ring or fused ring system substituent. The aryl group may be substituted or unsubstituted.
By xe2x80x9ccandidate compoundxe2x80x9d or xe2x80x9ctest compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is screened by employing one of the assay methods described herein. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, sugars, polysaccharides, and derivatives thereof.
By xe2x80x9cconditionxe2x80x9d is meant a state of being or feeling. Conditions include, but are not limited to a proliferative disease, for example, prostate cancer, breast cancer, gastrointestinal cancer, lung cancer, colon cancer, melanoma, ovarian cancer, gastric cancer, bladder cancer, salivary gland carcinoma, a brain tumor, leukemia, lymphoma, carcinoma, and the symptoms associated with cancer. Conditions also include myeloproliferative disorders.
By a xe2x80x9cdecreasexe2x80x9d is meant a lowering in the level of translation efficiency, as measured by a lowering in the level of reporter gene expression, as assayed, for example, by polysome analysis. The decrease is, for example, at least 10%, 30%, 40%, 50%, 75%, or 90% relative to a control sample that was not administered the compound, or that was contacted with the compound vehicle only.
By a xe2x80x9cderivativexe2x80x9d is meant a structural derivative having a chemical modification of the compound that enhances bioavailability, solubility, or stability in vivo or ex vivo, or that reduces the toxicity to non-target cells or dosage required. Such modifications are known to those skilled in the field of medicinal chemistry.
By a xe2x80x9cdosage sufficient to modulate HER2 polypeptide expressionxe2x80x9d is meant an amount of a chemical compound or small molecule that increases or decreases HER2 polypeptide expression when administered to a subject. For example, for a compound that decreases HER2 translation efficiency, the modulation is a decrease in HER2 polypeptide expression that is at least 10%, 30%, 40%, 50%, 75%, or 90% lower in a treated subject than in the same subject prior to the administration of the inhibitor or than in an untreated, control subject. In addition, for a compound that increases HER2 translation efficiency, the amount of HER2 polypeptide expression is, for example, at least 1.5-, 2-, 3-, 5-, 10-, or 20-fold greater in a treated subject than in the same subject prior to the administration of the modulator or than in an untreated, control subject.
By xe2x80x9cexposexe2x80x9d is meant to allow contact between an animal, cell, tissue, lysate or extract derived from a cell, or molecule derived from a cell, and a test compound.
By xe2x80x9cexpression vectorxe2x80x9d is meant a DNA construct that contains regulatory elements, for example, the UTR sequences of the present invention and a promoter that are operably linked to a downstream gene. Transfection of the expression vector into a recipient cell allows the cell to express RNA encoded by the expression vector. An expression vector may be a genetically engineered plasmid or virus, derived from, for example, a bacteriophage, adenovirus, retrovirus, poxvirus, herpesvirus, or artificial chromosome.
By an xe2x80x9cincreasexe2x80x9d is meant a rise in the level of translation efficiency, as measured by a rise in reporter gene expression, as assayed, for example, by polysome analysis. The increase is, for example, at least 1.5-fold to 2-fold, by at least 3-fold to 5-fold, or by at least 10-fold to 20-fold, relative to a control sample that was not administered the compound, or that was contacted with the compound vehicle only.
By xe2x80x9clower alkyl,xe2x80x9d xe2x80x9clower alkenyxe2x80x9d, and xe2x80x9clower alkynylxe2x80x9d is meant any carbon-containing radical having between 1 and 6 carbons, e.g., between 1 and 3 carbons or between 3 and 6 carbons, and only single, one or more double, or one or more triple carbon-carbon bonds, respectively. The radical may be substituted or unsubstituted. Exemplary xe2x80x9clowerxe2x80x9d groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, neopentyl, isopentyl, hexyl, 2-, 3-, or 4-methylpentyl, 2-ethylbutyl, and 2,2 or 3,3 dimethylbutyl.
By xe2x80x9cmodulatesxe2x80x9d is meant changing the level of translation efficiency, either by decrease or increase.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences. As used herein, an RNA comprising a regulatory element is operably linked to a promoter and/or 5xe2x80x2 UTR sequences and/or 3xe2x80x2 UTR sequences that direct transcription and/or translation of a reporter gene.
By xe2x80x9cpharmaceutically acceptable saltxe2x80x9d is meant a non-toxic salt of a compound of the invention formed, e.g., from non-toxic inorganic or organic acids. Such non-toxic salts include, for example, those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. Other pharmaceutically acceptable salts are known to those skilled in the art.
By xe2x80x9cproliferative diseasexe2x80x9d is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, bladder cancer, gastric cancer, salivary gland carcinoma, and lung cancer are all examples of proliferative disease. A myeloproliferative disease is another example of a proliferative disease.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription.
By xe2x80x9cproteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cpolypeptide fragmentxe2x80x9d is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
By xe2x80x9cregulatory elementxe2x80x9d is meant sequences that can modulate expression of a gene or gene product. Examples of regulatory sequences include, but are not limited to promoters, enhancers, sequences that stabilize an RNA sequence, sequences that enhance protein stability, translation termination sequences, and additional 5xe2x80x2 or 3xe2x80x2 UTR sequences.
By xe2x80x9creporter genexe2x80x9d or xe2x80x9creporter nucleic acidxe2x80x9d is meant any gene or translatable nucleotide sequence that encodes a product whose RNA or polypeptide expression is detectable and/or quantitatable by immunological, chemical, biochemical, or biological assays. A reporter gene may be detected at the nucleic acid level by detecting nucleic acid expression, for example, by Northern blot analysis, or filter binding assays. A reporter gene product may, for example, have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., xcex2-galactosidase, luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labeled antibody). It is understood that any engineered variants of reporter genes that are readily available to one skilled in the art, are also included, without restriction, in the foregoing definition. In addition, a reporter gene is any nucleic acid sequence that is not endogenously contained as an RNA sequence of the cell of interest.
By a xe2x80x9csubstantially pure nucleic acid,xe2x80x9d xe2x80x9cisolated nucleic acid,xe2x80x9d or xe2x80x9csubstantially pure and isolated nucleic acidxe2x80x9d is meant DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9ctherapeutically effective amountxe2x80x9d is meant an amount of a compound sufficient to produce a preventative, healing, curative, stabilizing, or ameliorative effect in the treatment of a condition, e.g., a proliferative disease.
By xe2x80x9ctransformed cellxe2x80x9d or xe2x80x9ctransfected cellxe2x80x9d is meant a cell (or a descendent of a cell) into which a nucleic acid molecule has been introduced, by means of recombinant nucleic acid techniques. Such cells may be either stably or transiently transfected.
By xe2x80x9ctransformationxe2x80x9d or xe2x80x9ctransfectionxe2x80x9d is meant any method for introducing foreign molecules into a cell (e.g., a bacterial, yeast, fungal, algal, plant, insect, or animal cell, particularly a mammalian cell). Lipofection, DEAE-dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the methods known to those skilled in the art which may be used. In addition, a foreign molecule can be introduced into a cell using a cell penetrating peptide, as described, for example, by Fawell et al. (Proc. Natl. Acad. Sci. USA 91:664-668 (1994)) and Lindgren et al. (TIPS 21:99-103 (2000)).
By xe2x80x9ctranslationxe2x80x9d is meant the process of generating a polypeptide that has an amino acid sequence dictated by the codon sequence of an mRNA that encodes the polypeptide.
By xe2x80x9ctranslation efficiencyxe2x80x9d is meant the ability of an RNA to be translated into a polypeptide. Translation efficiency can be measured, for example, by polysome distribution analysis, as described herein, where an RNA with a high level of translation efficiency will be associated with high molecular weight polysomes, and an RNA with a low level of translation efficiency will not be associated with high molecular weight polysomes, or will have a smaller amount associated with high molecular weight polysomes, or will be associated with low molecular weight polysomes. Alternatively, translation efficiency can be measured by determining the level of reporter coding protein expression or activity. This may be done in combination with the detection of steady state RNA levels to confirm that a modulation in reporter polypeptide expression or activity is a result of a change in RNA translation efficiency.
In addition, translation efficiency of HER2 can be detected by determining the viability of a cell contacted with a compound that modulates HER2 translation efficiency. Reduced HER2 protein expression in tumor cells has been shown to be associated with cell toxicity (Juhl et al., J. Biol. Chem. 272:29482-29486 (1997)). Therefore, a compound that decreases the translation efficiency of HER2 will result in increased cell death, compared to control cells that are not administered the compound, or that are contacted with the compound vehicle only. Methods for assaying cell death are well known in the art. For example, cell death can be measured by determining cellular ATP levels, wherein a cell that is undergoing cell death has a decreased level of cellular ATP compared to a control cell. Cell death may also be measured by staining with a vital dye, for example, trypan blue, wherein a cell that is dying will be stained with the vital dye, and a cell that is not dying will not be stained with the dye.
The level of RNA translation efficiency in a cell contacted with a compound that decreases the level of translation efficiency may be decreased, for example, by at least 10%, 30%, 40%, 50%, 75%, or 90% relative to a control cell that was not contacted with the compound, or that was contacted with the compound vehicle only. Alternatively, the level of RNA translation efficiency in a cell contacted with a compound that increases the level of translation efficiency may be increased, for example, by at least 1.5-fold to 2-fold, by at least 3-fold to 5-fold, or by at least 10-fold to 20-fold, relative to a control sample that was not administered the compound, or that was contacted with the compound vehicle only.
By xe2x80x9ctreatingxe2x80x9d is meant the medical management of a subject, e.g. an animal or human, with the intent that a prevention, cure, stabilization, or amelioration of the symptoms or condition will result. This term includes active treatment, that is, treatment directed specifically toward improvement of the disorder; palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disorder; preventive treatment, that is, treatment directed to prevention of disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disorder. The term xe2x80x9ctreatmentxe2x80x9d also includes symptomatic treatment, that is, treatment directed toward constitutional symptoms of the disorder. xe2x80x9cTreatingxe2x80x9d a condition with the compounds of the invention involves administering such a compound, alone or in combination and by any appropriate means, to an animal, cell, lysate or extract derived from a cell, or a molecule derived from a cell.
By xe2x80x9cunder conditions that allow interaction between a nucleic acid molecule and an RNA binding proteinxe2x80x9d is meant reaction conditions that permit a physical association between a nucleic acid molecule and a protein that is based on the specific characteristics of the interacting molecules, and is not inhibited by non-specific competitor molecules present at a concentration equivalent to the interacting molecules.
By a xe2x80x9cUTRxe2x80x9d is meant a nucleic acid sequence derived from the 5xe2x80x2 or 3xe2x80x2 untranslated region of a gene, wherein the sequence is composed of ten or more contiguous nucleotides, said nucleotides being outside of the coding region of a gene of interest. Preferably the UTR is the full length UTR sequence of a gene of interest, for example, the HER2 gene.